Ricin toxin (RT or ricin) is a naturally occurring toxin composed of an enzymatically active, cytotoxic "A" amino acid sequence, and a "B" sequence, which is presumed to be responsible both for attaching the "A" sequence to a target cell to be killed, and to aid in the translocation of A fragment into the cytoplasm. Other examples of such toxins include diphtheria toxin and the exotoxin from Pseudomonas aeruginosa. Other toxic proteins, such as, for example, those derived from Phytolacca americana (PAPI, PAPII, and PAP-S) and gelonin show in vitro activities comparable to the "A" sequences of the above toxins, but are inactive in vivo, presumably due to the absence of a "B" chain.
The "ricin" peptides of the present invention are derived from the seeds of Ricinus communis, commonly known as castor beans. Two similar proteins (often called lectins) are extractable from these seeds: the above-mentioned ricin and Ricin communis agglutinin (RCA). Both proteins contain A and B portions which do not comprise a single peptide but are joined by a disulfide link. The A portions of both ricin and RCA are capable of catalytically inactivating the large subunit of ribosomes in vitro and the mechanism of ricin for in vivo cytotoxicity is believed to reside in this capacity for ribosome inactivation. Ricin and RCA appear to be highly homologous but differences exist. RCA is dramatically less toxic, and appears to exhibit characteristics corresponding to those expected of a dimer of ricin.
Careful fractionation of castor bean extracts shows the presence of several ricin isotoxins. The distinction between ricins D and E has been previously disclosed (Mise, et al, Agric Biol Chem (1977) 41:2041-2046; Wei, et al, J Biol Chem (1978) 253:2061-2066: Lin, et al, Eur J Biochem (1980) 105:453-459; Genaud, et al, J Immunol Meth (1982) 49:323-332). Ricin D has a pI near 7.4 and a high affinity for agarose; ricin E has a pI near 8.8 and a low affinity for agarose. There are several reports of purported isotoxins which have been shown to be more acidic forms of ricin D (Olsnes, et al, J Biol Chem (1974) 249:803-810; Ishiguro, et al, Toxicon (1976) 14:157-165; Cawley, et al, Arch Biochem Biophys (1978) 190:744-755).
The differences in properties between ricins D and E seem to reside in the B chain (Funatsu, et al, Agric Biol Chem (1978) 42:851-859). The RTA chains from ricins D and E are identical in composition, pI, and apparent molecular weight. However, ricin D yields two distinct RTA species, RTA1 and RTA2. These isoenzymes differ in molecular weight by SDS-PAGE and in carbohydrate content, and can be resolved by ion exchange chromatography with a very shallow salt gradient (Olsnes, et al, Biochemistry (1973) 12:3121-3126).
U.S. patent application Ser. No. 747,114, filed Jun. 20, 1985, assigned to the same assignee and incorporated herein by reference discloses the separation of an additional and previously unreported ricin E isotoxin. For convenience, the ribotoxin most similar to the previous ricin E preparation was designated ricin El, and the novel ribotoxin was designated ricin E2. Ricin E2 has a pI identical to that of ricin E1. Compared to ricin E1, it is 1% as toxic to mice and 2-4% as toxic to cultured cell lines, is bound to agarose more tightly at moderate to high ionic strength, and is approximately 2 kD larger by SDS-PAGE.
The components of ricin and of RCA have been well characterized on the basis of the extracted materials, and their properties extensively reviewed: Olsnes, S., Perspectives in Toxicology, A. W. Bernheimer, Ed (1977) J. Wiley & Sons, NY, pp 122-147; Olsnes, S., et al, Molecular Action of Toxins and Viruses, Cohen, et al, Ed (1982) Elsevier, Amsterdam, pp 51-105. Ricin has an apparent molecular weight of 58,000 daltons and consists of the A chain with a molecular weight of 32,000 daltons and a B chain of molecular weight of 34,700 daltons. RCA is a tetramer which has two A subunits of molecular weight 32,000, and two B subunits of molecular weight 36,000 each. In their native environments, the A and B chains are generally glycosylated. The A and B subunits of both ricin and RCA are linked only by a single disulfide bond, and not by peptide linkage unlike, for example diphtheria toxin which is found as a single chain peptide. It is also known that both ricin and RCA, though having separate peptides for A and B portions, are each derived from a single chain precursor in each case (Butterworth, H. E., et al, Eur J Biochem (1983) 137:57). This precursor was shown to contain a sequence of 12 amino acids between the A chain (amino terminal) and B chain (carboxy terminal) sequence; U.S. Ser. No. 578,121, filed Feb. 8, 1984, assigned to the same assignee and incorporated herein by reference. The invention hereinbelow shows the ricin A sequence to contain 265 amino acids preceded by a 35 amino acid leader (signal) peptide. It is assumed that upon excision of the dodecameric intervening peptide, the A and B chains remain linked through the single disulfide bond.
With regard to the invention herein, three full-length ricin related clones have been isolated, two of which correspond to proteins of known sequence. The insert for pRT3 corresponds in the amino acid sequence encoded to the primary sequence of ricin agglutinin. The cDNA insert in pRT17 corresponds to the composite between the ricin toxin B chain encoded in the DNA disclosed in U.S. Ser. No. 578,121 (supra) and the ricin A encoding sequences described herein. This is the DNA, then, encoding the precursor for ricin D.
pRT38, on the other hand, encodes a new protein which, because of the predicted characteristics of the deduced protein in comparison to ricin D is presumed to be the DNA encoding ricin E. Specifically, ricin E has a pI considerably higher than that of ricin D, as disclosed above, therefore the deviations from homology which comprise changes from amino acids in ricin D neutral to basic amino acids in the new protein are consistent with this identification of the protein encoded.
The present invention provides a means for obtaining the A chain of ricin and the full length "precursor" chains of two ricin isotoxins and of RCA using recombinant technology. Native ricin A and native ricin exist in a number of homologous but not exactly identical forms depending on the plant variety used as source, but even protein derived from a single plant may exhibit more than one primary structure. Recombinantly produced ricin A, of course, permits production of a single desired amino acid sequence, and makes possible an exploration of the structural-features required for its activity. The techniques and materials of the present invention further permit selective modification of the amino acid sequence of the proteins and thus permit manipulation to provide properties which are capable of tailoring the cytotoxicity and other properties of these materials. The production of recombinant ricin B chain has been disclosed in U.S. Ser. No. 578,121 (supra). The invention herein, by enabling the production of ricin A and of full length ricin using predictable, efficient, and economic procedures which, further, permit directed modification, permits the use of these proteins in practical and improved ways not before possible. Further, by suitable recombinant manipulation employing, as well, the DNA sequence encoding B chain, the full length ricin toxin may be cloned and expressed and various hybrids containing portions of the several proteins may be obtained.
In addition, by using a novel construct employing codons for the leader sequence of a bacterial secreted protein, soluble biologically active ricin A chain and ricin precursor are directly obtained using procaryotic hosts, without need for further treatment to refold or solubilize the heterologous protein.